Preoperative portal vein embolization (PVE) is employed clinically to avoid postoperative liver insufficiency. Animal models are usually used to study PVE in terms of mechanisms and pathophysiological changes. PVE is formerly monitored by conventional absorption contrast imaging (ACI) with iodine contrast agent. However, the side effects induced by iodine can give rise to animal damage and death. In this study, the feasibility of using phase contrast imaging (PCI) to show PVE using homemade CO2 microbubbles in living rats has been investigated. CO2 gas was first formed from the reaction between citric acid and sodium bicarbonate. The CO2 gas was then encapsulated by egg white to fabricate CO2 microbubbles. ACI and PCI of CO2 microbubbles were performed and compared in vitro. An additional increase in contrast was detected in PCI. PCI showed that CO2 microbubbles gradually dissolved over time, and the remaining CO2 microbubbles became larger. By PCI, the CO2 microbubbles were found to have certain stability, suggesting their potential use as embolic agents. CO2 microbubbles were injected into the main portal trunk to perform PVE in living rats. PCI exploited the differences in the refractive index and facilitated clear visualization of the PVE after the injection of CO2 microbubbles. Findings from this study suggest that homemade CO2 microbubbles-based PCI is a novel modality for preclinical PVE research.